Isolation and Characterization of Novel Antibodies That Target the Blood-Brain Barrier

Tuesday, October 18, 2011: 4:15 PM
M100 H (Minneapolis Convention Center)
Charles C. Stutz1, Angela R. Jones2, Sue-Zanne Tan2 and Eric Shusta3, (1)Chemical and Biological Engineering, University of Wisconsin - Madison, Madison, WI, (2)University of Wisconsin - Madison, Madison, WI, (3)University of Wisconsin, Madison, WI

Central nervous system (CNS) disorders affect 1.5 billion people worldwide, and these numbers will continue to increase with an aging population.  Thus, it is important that CNS therapeutics continue to be developed and optimized.  One important challenge in the treatment of brain disease is the limitations on drug delivery imposed by the blood-brain barrier (BBB).  The BBB is a layer of tightly connected endothelial cells that prevents almost all molecules from entering the brain by diffusion.  In attempts to circumvent the BBB, the endogenous nutrient transporters present in the brain have been suggested as potential drug delivery portals.  The most studied of these transporters are the insulin receptor and transferrin receptor, both of which undergo receptor-mediated transcytosis (RMT).  Antibodies against these receptors have been conjugated with either drugs or tracers molecules and have been shown to penetrate the brain via the RMT pathway.  The drawback of using these two well-studied systems is that these transporters are also widely expressed elsewhere in the body.  This allows for significant accumulation of the antibody and payload in peripheral organs, which can both increase the risk of side effects and lowers the blood availability of the payload.   Thus, our goal has been to isolate novel RMT transporters and corresponding targeting antibodies that allow for more efficient and targeted drug delivery across the BBB.  To this end, a two-step combinatorial screen was performed using a phage-displayed library of human scFv.  In the first step, two rounds of in vitro endocytosis screening were performed on cultured primary rat brain endothelial cells or freshly isolated rat brain capillaries.  Prior to the internalization selection the libraries were pre-subtracted against peripheral endothelia to help promote BBB specificity.  The in vitro step serves to focus the antibody pool to clones that have the capacity to endocytose via RMT systems. The phage pools resulting from the second round of the in vitro selection were then used in in vivo transport screens (tail vein injection) and binding screens (transcardial perfusion). While the transport screens led to some promising antibody clones displaying BBB selectivity and brain uptake, the targeted receptors were only sporadically expressed throughout the brain.  In contrast, the binding screen substantially enriched the antibody pools for clones that bind the luminal side of the rat brain capillaries (14/27 clones tested). Promising phage-displayed antibody clones have been used in immunohistochemical staining of rat organ tissue sections to predict biodistribution, and BBB binding was confirmed by transcardial perfusion.   Several scFv have been also been produced as soluble proteins and are being used to test both in vivo biodistribution and to expression clone the cognate RMT system.  Taken together, these studies have resulted in a panel of BBB-targeted antibodies that could ultimately expand the repertoire of brain drug delivery options.

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See more of this Session: Protein Engineering III - Applications
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division